Rhotekin, an effector of Rho, is highly expressed in the brain but its function is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we raised the possibility that Rhotekin interacts with a PDZ-protein, PIST (PDZ domain protein interacting specifically with TC10) in vitro, and found that these proteins form complex in the rat brain tissues. We then demonstrated that Rhotekin and PIST are expressed in developmental stage-specific manners in the rat brain. In immunofluorescence analyses, Rhotekin and PIST were suggested to co-localize at synapses in rat primary cultured hippocampal neurons. On the other hand, PIST was found to form immunocomplex with another PDZ-binding protein, beta-catenin, in HEK293 cells and in the rat brain, and co-localized with this protein at dendritic filopods. The interaction of PIST with beta-catenin was inhibited by the presence of Rhotekin. These results suggest a possible yet unidentified role of Rhotekin, in harmony with PIST and beta-catenin, in neuronal cells.